Chimeric aquaretic and natriuretic polypeptides lacking vasodilatory activity

ABSTRACT

This document provides aquaretic and natriuretic polypeptides. For example, this document provides polypeptides having aquaretic and/or natriuretic activities. In some cases, a polypeptide provided herein can have aquaretic and natriuretic activities, while lacking the ability to lower blood pressure. This document also provides methods and materials for inducing aquaretic and/or natriuretic activities within a mammal.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/592,819, filed Aug. 23, 2012, which is a divisional of U.S.application Ser. No. 12/440,053, filed Apr. 12, 2010, now U.S. Pat. No.8,283,318, which is a National Stage application under 35 U.S.C. §371 ofInternational Application No. PCT/US2007/077900, having an internationalfiling date of Sep. 7, 2007, which claims benefit of U.S. ProvisionalApplication No. 60/825,028, filed Sep. 8, 2006.

BACKGROUND

1. Technical Field

This document relates to aquaretic and natriuretic polypeptides. Forexample, this document relates to polypeptides having aquaretic andnatriuretic activities while lacking the ability dilate vascular tissue.

2. Background Information

Members of the natriuretic polypeptide family are hormones that regulatebody fluid homeostasis. Atrial natriuretic peptide (ANP) is secreted byatrial myocytes in response to increased intravascular volume. Once ANPis in the circulation, its effects are primarily on the kidney, vasculartissue, and adrenal gland, in which its actions lead to the excretion ofsodium and water by the kidneys and a decrease in intravascular volumeand blood pressure. BNP also is of myocardial cell origin, and like ANP,it circulates in human plasma. BNP is natriuretic, rennin inhibiting,vasodilating, and lusitropic. The main circulating and storage form ofBNP is a 32 amino acid polypeptide with a ring structure. Physiologicalactions of BNP are mediated through a guanylate cyclase-linked receptor,natriuretic peptide receptor A (NPR-A). Clearance of BNP is promoted bya NPR-C receptor that removes it from the circulation. BNP also isdegraded through enzymatic cleavage by neutral endopeptidase. C-typenatriuretic peptide (CNP) is of endothelial cell origin and functions asa vasodilating and growth-inhibiting polypeptide. Dendroaspisnatriuretic peptide (DNP) is similar in structure to ANP, BNP, and CNP,and is isolated from the venom of Dendroaspis angusticeps or green mambasnake.

SUMMARY

This document relates to aquaretic and natriuretic polypeptides. Forexample, this document provides polypeptides having aquaretic andnatriuretic activities. In some cases, a polypeptide provided herein canhave aquaretic and natriuretic activities, while lacking the ability todilate vascular tissue.

In general, one aspect of this document features a polypeptide less than45 amino acid residues in length, wherein the polypeptide comprises, orconsists essentially of, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:1 or the sequence setforth in SEQ ID NO:1 with no more than three mismatches, (b) thesequence set forth in SEQ ID NO:2 or the sequence set forth in SEQ IDNO:2 with no more than eight mismatches, and (c) the sequence set forthin SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with no morethan three mismatches. The polypeptide can comprise natriureticactivity. The polypeptide can lack vasodilatory activity. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1, thesequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ IDNO:3. The polypeptide can be a substantially pure polypeptide.

In another embodiment, this document features an isolated nucleic acidencoding a polypeptide less than 45 amino acid residues in length,wherein the polypeptide comprises, or consists essentially of, in anorder from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 with nomore than three mismatches, (b) the sequence set forth in SEQ ID NO:2 orthe sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can comprise the sequenceset forth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a vector comprising, orconsisting essentially of, a nucleic acid encoding a polypeptide lessthan 45 amino acid residues in length, wherein the polypeptidecomprises, or consists essentially of, in an order from amino terminusto carboxy terminus: (a) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than three mismatches,(b) the sequence set forth in SEQ ID NO:2 or the sequence set forth inSEQ ID NO:2 with no more than eight mismatches, and (c) the sequence setforth in SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with nomore than three mismatches. The polypeptide can comprise natriureticactivity. The polypeptide can lack vasodilatory activity. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1, thesequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ IDNO:3. The polypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a host cell comprising anucleic acid encoding a polypeptide less than 45 amino acid residues inlength, wherein the polypeptide comprises, or consists essentially of,in an order from amino terminus to carboxy terminus: (a) the sequenceset forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 withno more than three mismatches, (b) the sequence set forth in SEQ ID NO:2or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can comprise the sequenceset forth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide. The host cell can be a eukaryotic hostcell.

In another embodiment, this document features a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apolypeptide less than 45 amino acid residues in length, wherein thepolypeptide comprises, or consists essentially of, in an order fromamino terminus to carboxy terminus: (a) the sequence set forth in SEQ IDNO:1 or the sequence set forth in SEQ ID NO:1 with no more than threemismatches, (b) the sequence set forth in SEQ ID NO:2 or the sequenceset forth in SEQ ID NO:2 with no more than eight mismatches, and (c) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can lack vasodilatory activity.The polypeptide can comprise the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:1, thesequence set forth in SEQ ID NO:2, and the sequence set forth in SEQ IDNO:3. The polypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a method for increasingaquaretic and natriuretic activity within a mammal without loweringblood pressure, wherein the method comprises administering, to themammal, a polypeptide less than 45 amino acid residues in length,wherein the polypeptide comprises, or consists essentially of, in anorder from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 with nomore than three mismatches, (b) the sequence set forth in SEQ ID NO:2 orthe sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can comprise the sequenceset forth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a stent comprising acoating comprising a polypeptide less than 45 amino acid residues inlength, wherein the polypeptide comprises, or consists essentially of,in an order from amino terminus to carboxy terminus: (a) the sequenceset forth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 withno more than three mismatches, (b) the sequence set forth in SEQ ID NO:2or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can comprise the sequenceset forth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a dialysis tubingcomprising a coating comprising a polypeptide less than 45 amino acidresidues in length, wherein the polypeptide comprises, or consistsessentially of, in an order from amino terminus to carboxy terminus: (a)the sequence set forth in SEQ ID NO:1 or the sequence set forth in SEQID NO:1 with no more than three mismatches, (b) the sequence set forthin SEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with no morethan eight mismatches, and (c) the sequence set forth in SEQ ID NO:3 orthe sequence set forth in SEQ ID NO:3 with no more than threemismatches. The polypeptide can comprise natriuretic activity. Thepolypeptide can lack vasodilatory activity. The polypeptide can comprisethe sequence set forth in SEQ ID NO:1. The polypeptide can comprise thesequence set forth in SEQ ID NO:2. The polypeptide can comprise thesequence set forth in SEQ ID NO:3. The polypeptide can comprise thesequence set forth in SEQ ID NO:1, the sequence set forth in SEQ IDNO:2, and the sequence set forth in SEQ ID NO:3. The polypeptide can bea substantially pure polypeptide.

In another embodiment, this document features microparticles ornanoparticles comprising a polypeptide less than 45 amino acid residuesin length, wherein the polypeptide comprises, or consists essentiallyof, in an order from amino terminus to carboxy terminus: (a) thesequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ IDNO:1 with no more than three mismatches, (b) the sequence set forth inSEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with no more thaneight mismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can comprise the sequenceset forth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a polypeptide less than 45amino acid residues in length, wherein the polypeptide comprises, in anorder from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:1 or the sequence set forth in SEQ ID NO:1 with nomore than three mismatches, (b) the sequence set forth in SEQ ID NO:7 orthe sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide cancomprise the sequence set forth in SEQ ID NO:1. The polypeptide cancomprise the sequence set forth in SEQ ID NO:7. The polypeptide cancomprise the sequence set forth in SEQ ID NO:3. The polypeptide cancomprise the sequence set forth in SEQ ID NO:1, the sequence set forthin SEQ ID NO:7, and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

In another embodiment, this document features an isolated nucleic acidencoding a polypeptide less than 45 amino acid residues in length,wherein the polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:1 or thesequence set forth in SEQ ID NO:1 with no more than three mismatches,(b) the sequence set forth in SEQ ID NO:7 or the sequence set forth inSEQ ID NO:2 with no more than eight mismatches, and (c) the sequence setforth in SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with nomore than three mismatches. The polypeptide can comprise natriureticactivity. The polypeptide can comprise the sequence set forth in SEQ IDNO:1. The polypeptide can comprise the sequence set forth in SEQ IDNO:7. The polypeptide can comprise the sequence set forth in SEQ IDNO:3. The polypeptide can comprise the sequence set forth in SEQ IDNO:1, the sequence set forth in SEQ ID NO:7, and the sequence set forthin SEQ ID NO:3. The polypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a vector comprising, orconsisting essentially of, a nucleic acid encoding a polypeptide lessthan 45 amino acid residues in length, wherein the polypeptidecomprises, in an order from amino terminus to carboxy terminus: (a) thesequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ IDNO:1 with no more than three mismatches, (b) the sequence set forth inSEQ ID NO:7 or the sequence set forth in SEQ ID NO:2 with no more thaneight mismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide cancomprise the sequence set forth in SEQ ID NO:1. The polypeptide cancomprise the sequence set forth in SEQ ID NO:7. The polypeptide cancomprise the sequence set forth in SEQ ID NO:3. The polypeptide cancomprise the sequence set forth in SEQ ID NO:1, the sequence set forthin SEQ ID NO:7, and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a host cell comprising anucleic acid encoding a polypeptide less than 45 amino acid residues inlength, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:1or the sequence set forth in SEQ ID NO:1 with no more than threemismatches, (b) the sequence set forth in SEQ ID NO:7 or the sequenceset forth in SEQ ID NO:2 with no more than eight mismatches, and (c) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:7, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apolypeptide less than 45 amino acid residues in length, wherein thepolypeptide comprises, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:1 or the sequence setforth in SEQ ID NO:1 with no more than three mismatches, (b) thesequence set forth in SEQ ID NO:7 or the sequence set forth in SEQ IDNO:2 with no more than eight mismatches, and (c) the sequence set forthin SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with no morethan three mismatches. The polypeptide can comprise natriureticactivity. The polypeptide can comprise the sequence set forth in SEQ IDNO:1. The polypeptide can comprise the sequence set forth in SEQ IDNO:7. The polypeptide can comprise the sequence set forth in SEQ IDNO:3. The polypeptide can comprise the sequence set forth in SEQ IDNO:1, the sequence set forth in SEQ ID NO:7, and the sequence set forthin SEQ ID NO:3. The polypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a stent comprising acoating comprising a polypeptide less than 45 amino acid residues inlength, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:1or the sequence set forth in SEQ ID NO:1 with no more than threemismatches, (b) the sequence set forth in SEQ ID NO:7 or the sequenceset forth in SEQ ID NO:2 with no more than eight mismatches, and (c) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:7, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a dialysis tubingcomprising a coating comprising a polypeptide less than 45 amino acidresidues in length, wherein the polypeptide comprises, in an order fromamino terminus to carboxy terminus: (a) the sequence set forth in SEQ IDNO:1 or the sequence set forth in SEQ ID NO:1 with no more than threemismatches, (b) the sequence set forth in SEQ ID NO:7 or the sequenceset forth in SEQ ID NO:2 with no more than eight mismatches, and (c) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:7, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features microparticles ornanoparticles comprising a polypeptide less than 45 amino acid residuesin length, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:1or the sequence set forth in SEQ ID NO:1 with no more than threemismatches, (b) the sequence set forth in SEQ ID NO:7 or the sequenceset forth in SEQ ID NO:2 with no more than eight mismatches, and (c) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:7, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a polypeptide less than 45amino acid residues in length, wherein the polypeptide comprises, in anorder from amino terminus to carboxy terminus: (a) the sequence setforth in SEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with nomore than eight mismatches, and (b) the sequence set forth in SEQ IDNO:3 or the sequence set forth in SEQ ID NO:3 with no more than threemismatches. The polypeptide can comprise natriuretic activity. Thepolypeptide can lack vasodilatory activity. The polypeptide can lack thesequence set forth in SEQ ID NO:1. The polypeptide can comprise thesequence set forth in SEQ ID NO:2. The polypeptide can comprise thesequence set forth in SEQ ID NO:3. The polypeptide can comprise thesequence set forth in SEQ ID NO:2 and the sequence set forth in SEQ IDNO:3. The polypeptide can be a substantially pure polypeptide.

In another embodiment, this document features an isolated nucleic acidencoding a polypeptide less than 45 amino acid residues in length,wherein the polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:2 or thesequence set forth in SEQ ID NO:2 with no more than eight mismatches,and (b) the sequence set forth in SEQ ID NO:3 or the sequence set forthin SEQ ID NO:3 with no more than three mismatches. The polypeptide cancomprise natriuretic activity. The polypeptide can lack vasodilatoryactivity. The polypeptide can lack the sequence set forth in SEQ IDNO:1. The polypeptide can comprise the sequence set forth in SEQ IDNO:2. The polypeptide can comprise the sequence set forth in SEQ IDNO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO:2and the sequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a vector comprising, orconsisting essentially of, a nucleic acid encoding a polypeptide lessthan 45 amino acid residues in length, wherein the polypeptidecomprises, in an order from amino terminus to carboxy terminus: (a) thesequence set forth in SEQ ID NO:2 or the sequence set forth in SEQ IDNO:2 with no more than eight mismatches, and (b) the sequence set forthin SEQ ID NO:3 or the sequence set forth in SEQ ID NO:3 with no morethan three mismatches. The polypeptide can comprise natriureticactivity. The polypeptide can lack vasodilatory activity. Thepolypeptide can lack the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2 and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a host cell comprising anucleic acid encoding a polypeptide less than 45 amino acid residues inlength, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:2or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (b) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can lack the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:2 and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apolypeptide less than 45 amino acid residues in length, wherein thepolypeptide comprises, in an order from amino terminus to carboxyterminus: (a) the sequence set forth in SEQ ID NO:2 or the sequence setforth in SEQ ID NO:2 with no more than eight mismatches, and (b) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can lack vasodilatory activity.The polypeptide can lack the sequence set forth in SEQ ID NO:1. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:3. Thepolypeptide can comprise the sequence set forth in SEQ ID NO:2 and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a method for increasingaquaretic and natriuretic activity within a mammal without loweringblood pressure, wherein the method comprises administering, to themammal, a polypeptide less than 45 amino acid residues in length,wherein the polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:2 or thesequence set forth in SEQ ID NO:2 with no more than eight mismatches,and (b) the sequence set forth in SEQ ID NO:3 or the sequence set forthin SEQ ID NO:3 with no more than three mismatches. The polypeptide cancomprise natriuretic activity. The polypeptide can lack vasodilatoryactivity. The polypeptide can lack the sequence set forth in SEQ IDNO:1. The polypeptide can comprise the sequence set forth in SEQ IDNO:2. The polypeptide can comprise the sequence set forth in SEQ IDNO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO:2and the sequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a method for increasingaquaretic and natriuretic activity within a mammal without loweringblood pressure, wherein the method comprises administering, to themammal, a polypeptide less than 45 amino acid residues in length,wherein the polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:2 or thesequence set forth in SEQ ID NO:2 with no more than eight mismatches,and (b) the sequence set forth in SEQ ID NO:3 or the sequence set forthin SEQ ID NO:3 with no more than three mismatches. The polypeptide cancomprise natriuretic activity. The polypeptide can lack vasodilatoryactivity. The polypeptide can lack the sequence set forth in SEQ IDNO:1. The polypeptide can comprise the sequence set forth in SEQ IDNO:2. The polypeptide can comprise the sequence set forth in SEQ IDNO:3. The polypeptide can comprise the sequence set forth in SEQ ID NO:2and the sequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In another embodiment, this document features a stent comprising acoating comprising a polypeptide less than 45 amino acid residues inlength, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:2or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (b) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can lack the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:2 and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

In another embodiment, this document features a dialysis tubingcomprising a coating comprising a polypeptide less than 45 amino acidresidues in length, wherein the polypeptide comprises, in an order fromamino terminus to carboxy terminus: (a) the sequence set forth in SEQ IDNO:2 or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (b) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can lack the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:2 and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

In another embodiment, this document features microparticles ornanoparticles comprising a polypeptide less than 45 amino acid residuesin length, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:2or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (b) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can lack the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:2 and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

In another embodiment, this document features method for treating amammal having a cardiovascular or renal condition. The method cancomprise administering a polypeptide to the mammal. The mammal can havecardiovascular disease. The mammal can have congestive heart failure.The mammal can have myocardial infarction, a coronary disease, an arterydisease, a renal insufficiency, cancer, or a sodium and water retainingstate.

In some cases, the polypeptide can be less than 45 amino acid residuesin length, wherein the polypeptide comprises, or consists essentiallyof, in an order from amino terminus to carboxy terminus: (a) thesequence set forth in SEQ ID NO:1 or the sequence set forth in SEQ IDNO:1 with no more than three mismatches, (b) the sequence set forth inSEQ ID NO:2 or the sequence set forth in SEQ ID NO:2 with no more thaneight mismatches, and (c) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can comprise the sequenceset forth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:2, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In some cases, the polypeptide can be less than 45 amino acid residuesin length, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:1or the sequence set forth in SEQ ID NO:1 with no more than threemismatches, (b) the sequence set forth in SEQ ID NO:7 or the sequenceset forth in SEQ ID NO:2 with no more than eight mismatches, and (c) thesequence set forth in SEQ ID NO:3 or the sequence set forth in SEQ IDNO:3 with no more than three mismatches. The polypeptide can comprisenatriuretic activity. The polypeptide can comprise the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:7. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:1, the sequence set forth in SEQ ID NO:7, and thesequence set forth in SEQ ID NO:3. The polypeptide can be asubstantially pure polypeptide.

In some cases, the polypeptide can be less than 45 amino acid residuesin length, wherein the polypeptide comprises, in an order from aminoterminus to carboxy terminus: (a) the sequence set forth in SEQ ID NO:2or the sequence set forth in SEQ ID NO:2 with no more than eightmismatches, and (b) the sequence set forth in SEQ ID NO:3 or thesequence set forth in SEQ ID NO:3 with no more than three mismatches.The polypeptide can comprise natriuretic activity. The polypeptide canlack vasodilatory activity. The polypeptide can lack the sequence setforth in SEQ ID NO:1. The polypeptide can comprise the sequence setforth in SEQ ID NO:2. The polypeptide can comprise the sequence setforth in SEQ ID NO:3. The polypeptide can comprise the sequence setforth in SEQ ID NO:2 and the sequence set forth in SEQ ID NO:3. Thepolypeptide can be a substantially pure polypeptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of an ABC-NP polypeptide that is 33 aminoacid residues in length (SEQ ID NO:4). The first five amino acidresidues of SEQ ID NO:4 correspond to amino acid residues 11 to 15 ofhuman mature ANP and are designated as SEQ ID NO:1. Amino acid residues6 to 27 of SEQ ID NO:4 correspond to amino acid residues 1 to 22 ofhuman mature CNP with the exception that the amino acid residues atpositions 15, 16, and 17 are changed to arginine, glutamic acid, andalanine. Amino acid residues 6 to 27 of SEQ ID NO:4 are designated asSEQ ID NO:2. Amino acid residues 28 to 33 of SEQ ID NO:4 correspond toamino acid residues 27 to 32 of human mature BNP and are designated asSEQ ID NO:3.

FIG. 2 is a schematic diagram of an ABC-NP1 polypeptide that is 33 aminoacid residues in length (SEQ ID NO:6). The first five amino acidresidues of SEQ ID NO:6 correspond to amino acid residues 11 to 15 ofhuman mature ANP and are designated as SEQ ID NO:1. Amino acid residues6 to 27 of SEQ ID NO:6 correspond to amino acid residues 1 to 22 ofhuman mature CNP and are designated as SEQ ID NO:7. Amino acid residues28 to 33 of SEQ ID NO:6 correspond to amino acid residues 27 to 32 ofhuman mature BNP and are designated as SEQ ID NO:3.

FIG. 3 is a schematic diagram of a BC-NP2 polypeptide that is 28 aminoacid residues in length (SEQ ID NO:8). Amino acid residues 1 to 22 ofSEQ ID NO:8 correspond to amino acid residues 1 to 22 of human matureCNP with the exception that the amino acid residues at positions 15, 16,and 17 are changed to arginine, glutamic acid, and alanine. Amino acidresidues 1 to 22 of SEQ ID NO:8 are designated as SEQ ID NO:2. Aminoacid residues 23 to 28 of SEQ ID NO:8 correspond to amino acid residues27 to 32 of human mature BNP and are designated as SEQ ID NO:3.

FIG. 4 contains four bar graphs plotting urinary sodium excretion ratesfor dogs treated with ABC-NP, BNP, ABC-NP, or BC-NP2 (25 μg per kgadministered as an IV bolus). Baseline is prior to administration,whereas the indicated times are post administration.

FIG. 5 contains four bar graphs plotting urine flow rates for dogstreated with ABC-NP, BNP, ABC-NP, or BC-NP2 (25 μg per kg administeredas an IV bolus). Baseline is prior to administration, whereas theindicated times are post administration.

FIG. 6 contains four bar graphs plotting mean arterial blood pressurelevels for dogs treated with ABC-NP, BNP, ABC-NP, or BC-NP2 (25 μg perkg administered as an IV bolus). Baseline is prior to administration,whereas the indicated times are post administration.

FIG. 7 contains four bar graphs plotting plasma vasopressin levels fordogs treated with ABC-NP, BNP, ABC-NP, or BC-NP2 (25 μg per kgadministered as an IV bolus). Baseline is prior to administration,whereas the indicated times are post administration.

FIG. 8 contains four bar graphs plotting renal blood flow for dogstreated with ABC-NP, BNP, ABC-NP, or BC-NP2 (25 μg per kg administeredas an IV bolus). Baseline is prior to administration, whereas theindicated times are post administration.

FIG. 9 is a bar graph plotting the level of cGMP (pmol/mL) in untreatedhuman cardiac fibroblasts and human cardiac fibroblasts treated with ANP(10⁻⁶ M), BNP (10⁻⁶ M), CNP (10⁻⁶ M), ABC-NP (10⁻¹¹ M, 10⁻⁸ M, or 10⁻⁶M), or ABC-NP+BNP (10⁻⁶ M each) for 10 minutes.

FIG. 10 is a bar graph plotting the level of cGMP (pmol/mL) in untreatedhuman cardiac fibroblasts and human cardiac fibroblasts treated with ANP(10⁻⁶ M), BNP (10⁻⁶ M), CNP (10⁻⁶ M), ABC-NP1 (10⁻¹¹ M, 10⁻⁸ M, or 10⁻⁶M), ABC-NP1+BNP (10⁻⁶ M each) for 10 minutes.

FIG. 11 is a bar graph plotting the level of cGMP (pmol/mL) in untreatedhuman cardiac fibroblasts and human cardiac fibroblasts treated with ANP(10⁻⁶ M), BNP (10⁻⁶ M), CNP (10⁻⁶ M), or BC-NP2 (10⁻¹¹ M, 10⁻⁸ M, or10⁻⁶ M) for 10 minutes.

FIG. 12 contains a nucleic acid sequence (SEQ ID NO:5) that can encodean ABC-NP polypeptide having the amino acid sequence set forth in SEQ IDNO:4.

DETAILED DESCRIPTION

This document relates to aquaretic and natriuretic polypeptides. Forexample, this document provides polypeptides having aquaretic and/ornatriuretic activities. In some cases, a polypeptide provided herein canhave aquaretic and natriuretic activities, while lacking the ability tolower blood pressure. This document also provides methods and materialsfor inducing aquaretic and/or natriuretic activities within a mammal.

A polypeptide provided herein can have any sequence and can have anylength. For example, a polypeptide provided herein can include thesequence set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In somecases, a polypeptide provided herein can contain an amino acid sequencethat aligns to (a) the sequence set forth in SEQ ID NO:1 with four orless (e.g., three or less, two or less, one, or zero) amino acidadditions, deletions, substitutions, or combinations thereof, (b) thesequence set forth in SEQ ID NO:2 with ten or less (e.g., nine or less,eight or less, seven or less, six or less, five or less, four or less,three or less, two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof and (c) the sequenceset forth in SEQ ID NO:3 with five or less (e.g., four or less, three orless, two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof. For example, a polypeptideprovided herein can contain the sequence set forth in SEQ ID NO:1 withthe exception that the first arginine residue or the last isoleucineresidue of SEQ ID NO:1 is deleted or replaced with a different aminoacid residue.

In some cases, a polypeptide provided herein can contain (a) a firstamino acid sequence that either is set forth in SEQ ID NO:1 or aligns tothe sequence set forth in SEQ ID NO:1 with four or less (e.g., three orless, two or less, one, or zero) amino acid deletions, substitutions, orcombinations thereof, (b) a second amino acid sequence that either isset forth in SEQ ID NO:2 or aligns to the sequence set forth in SEQ IDNO:2 with ten or less (e.g., nine or less, eight or less, seven or less,six or less, five or less, four or less, three or less, two or less,one, or zero) amino acid additions, substitutions, or combinationsthereof, and (a) a third amino acid sequence that either is set forth inSEQ ID NO:3 or aligns to the sequence set forth in SEQ ID NO:3 with fiveor less (e.g., four or less, three or less, two or less, one, or zero)amino acid deletions, substitutions, or combinations thereof. Forexample, a polypeptide provided herein can comprise or consist of thesequence set forth in SEQ ID NO:4.

In some cases, a polypeptide provided herein can include the sequenceset forth in SEQ ID NO:1, SEQ ID NO:7, and SEQ ID NO:3. In some cases, apolypeptide provided herein can contain an amino acid sequence thataligns to (a) the sequence set forth in SEQ ID NO:1 with four or less(e.g., three or less, two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof, (b) the sequence setforth in SEQ ID NO:7 with ten or less (e.g., nine or less, eight orless, seven or less, six or less, five or less, four or less, three orless, two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof and (c) the sequence set forth inSEQ ID NO:3 with five or less (e.g., four or less, three or less, two orless, one, or zero) amino acid additions, deletions, substitutions, orcombinations thereof. For example, a polypeptide provided herein cancontain the sequence set forth in SEQ ID NO:1 with the exception thatthe first arginine residue or the last isoleucine residue of SEQ ID NO:1is deleted or replaced with a different amino acid residue.

In some cases, a polypeptide provided herein can contain (a) a firstamino acid sequence that either is set forth in SEQ ID NO:1 or aligns tothe sequence set forth in SEQ ID NO:1 with four or less (e.g., three orless, two or less, one, or zero) amino acid deletions, substitutions, orcombinations thereof, (b) a second amino acid sequence that either isset forth in SEQ ID NO:7 or aligns to the sequence set forth in SEQ IDNO:7 with ten or less (e.g., nine or less, eight or less, seven or less,six or less, five or less, four or less, three or less, two or less,one, or zero) amino acid additions, substitutions, or combinationsthereof, and (a) a third amino acid sequence that either is set forth inSEQ ID NO:3 or aligns to the sequence set forth in SEQ ID NO:3 with fiveor less (e.g., four or less, three or less, two or less, one, or zero)amino acid deletions, substitutions, or combinations thereof. Forexample, a polypeptide provided herein can comprise or consist of thesequence set forth in SEQ ID NO:6.

In some cases, a polypeptide provided herein can include the sequenceset forth in SEQ ID NO:2 and SEQ ID NO:3. In some cases, a polypeptideprovided herein can contain an amino acid sequence that aligns to (a)the sequence set forth in SEQ ID NO:2 with ten or less (e.g., nine orless, eight or less, seven or less, six or less, five or less, four orless, three or less, two or less, one, or zero) amino acid additions,deletions, substitutions, or combinations thereof and (b) the sequenceset forth in SEQ ID NO:3 with five or less (e.g., four or less, three orless, two or less, one, or zero) amino acid additions, deletions,substitutions, or combinations thereof. For example, a polypeptideprovided herein can contain the sequence set forth in SEQ ID NO:2 withthe exception that the first glycine residue of SEQ ID NO:2 is deletedor replaced with a different amino acid residue.

In some cases, a polypeptide provided herein can contain (a) a firstamino acid sequence that either is set forth in SEQ ID NO:2 or aligns tothe sequence set forth in SEQ ID NO:2 with ten or less (e.g., nine orless, eight or less, seven or less, six or less, five or less, four orless, three or less, two or less, one, or zero) amino acid additions,substitutions, or combinations thereof and (b) a second amino acidsequence that either is set forth in SEQ ID NO:3 or aligns to thesequence set forth in SEQ ID NO:3 with five or less (e.g., four or less,three or less, two or less, one, or zero) amino acid deletions,substitutions, or combinations thereof. For example, a polypeptideprovided herein can comprise or consist of the sequence set forth in SEQID NO:8.

A polypeptide provided herein can have any length. For example, apolypeptide provided herein can be between 25 and 45 (e.g., between 26and 44, between 27 and 43, between 28 and 42, between 29 and 41, between30 and 40, between 31 and 39, or between 32 and 38) amino acid residuesin length. It will be appreciated that a polypeptide with a length of 25or 45 amino acid residues is a polypeptide with a length between 25 and45 amino acid residues.

In some cases, a polypeptide provided herein can be a substantially purepolypeptide. As used herein, the term “substantially pure” withreference to a polypeptide means that the polypeptide is substantiallyfree of other polypeptides, lipids, carbohydrates, and nucleic acid withwhich it is naturally associated. Thus, a substantially pure polypeptideis any polypeptide that is removed from its natural environment and isat least 60 percent pure or is any chemically synthesized polypeptide. Asubstantially pure polypeptide can be at least about 60, 65, 70, 75, 80,85, 90, 95, or 99 percent pure. Typically, a substantially purepolypeptide will yield a single major band on a non-reducingpolyacrylamide gel.

In some embodiments, a polypeptide provided herein can lackvasoactivity. Vasoactivity can be assessed by determining responsitivityof a blood vessel (e.g., a carotid artery in an organ chamber) to thepolypeptide.

A polypeptide provide herein can be obtained by expression of arecombinant nucleic acid encoding the polypeptide or by chemicalsynthesis. For example, standard recombinant technology using expressionvectors encoding a polypeptide provide herein can be used. The resultingpolypeptides then can be purified using, for example, affinitychromatographic techniques and HPLC. The extent of purification can bemeasured by any appropriate method, including but not limited to: columnchromatography, polyacrylamide gel electrophoresis, or high-performanceliquid chromatography. A polypeptide provide herein can be designed orengineered to contain a tag sequence that allows the polypeptide to bepurified (e.g., captured onto an affinity matrix). For example, a tagsuch as c-myc, hemagglutinin, polyhistidine, or Flag™ tag (Kodak) can beused to aid polypeptide purification. Such tags can be inserted anywherewithin the polypeptide including at either the carboxyl or aminotermini. Other fusions that can be used include enzymes that aid in thedetection of the polypeptide, such as alkaline phosphatase.

A polypeptide provided herein can be produced to contain three regions,a first region that includes an N-terminus (e.g., an N-terminus sequencefrom an ANP polypeptide), a second region that includes a ring structureof a mature natriuretic polypeptide such as a CNP polypeptide, and athird region that includes a C-terminus (e.g., a C-terminus sequencefrom a BNP polypeptide). The N-termini, ring structures, and C-terminiof BNP, DNP, ANP, and CNP are described elsewhere. See, e.g., U.S.patent application Ser. No. 10/561,014.

A polypeptide provided herein can be used to treat cardiovasculardiseases, congestive heart failure, myocardial infarction, coronaryartery diseases, renal diseases, anti-ischemic renal diseases,anti-inflammatory renal diseases, anti-fibrotic renal diseases, hepaticdiseases, cancer (e.g., adenocarcinoma, squamous cell cancer, small celllung cancer, or breast cancer), pulmonary hypertension, vasculardiseases, diastolic dysfunction, cardiac dysfunction, renalinsufficiency (e.g., contrast induced or ischemic induced renalinsufficiency) or combinations thereof. For example, an ABC-NPpolypeptide can be administered to a human having coronary arterydisease under conditions wherein the severity of the human's coronaryartery disease symptoms is reduced.

A polypeptide provided herein can be formulated as a pharmaceuticalcomposition by admixture with pharmaceutically acceptable non-toxicexcipients or carriers. Such compositions can be administered to asubject in need thereof in an amount effective to treat, for example,heart, liver, vascular, kidney, or other sodium retaining conditions.Pharmaceutical compositions may be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions in aqueous physiological buffer solutions; for oraladministration, particularly in the form of tablets or capsules; or forintranasal administration, particularly in the form of powders, nasaldrops, or aerosols. Compositions for other routes of administration maybe prepared as desired using standard methods.

Formulations for parenteral administration may contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenatednaphthalenes, and the like. In particular, biocompatible, biodegradablelactide polymer, lactide/glycolide copolymer, orpolyoxethylene-polyoxypropylene copolymers are examples of excipientsfor controlling the release of the polypeptide in vivo. Other suitableparenteral delivery systems include ethylene-vinyl acetate copolymerparticles, osmotic pumps, implantable infusion systems, and liposomes.Formulations for inhalation administration may contain excipients suchas lactose, if desired. Inhalation formulations may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or they may be oily solutions for administration inthe form of nasal drops. If desired, the compounds can be formulated asgels to be applied intranasally. Formulations for parenteraladministration may also include glycocholate for buccal administration

For oral administration, tablets or capsules can be prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g. magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). Tablets can be coated by methods known in the art.Preparations for oral administration can also be formulated to givecontrolled release of the compound.

Nasal preparations can be presented in a liquid form or as a dryproduct. Nebulized aqueous suspensions or solutions can include carriersor excipients to adjust pH and/or tonicity.

In some cases, a polypeptide provided herein can be formulated as asustained release dosage form. For example, an ABC-NP, ABC-NP1, orBC-NP2 polypeptide can be formulated into a controlled releaseformulation. In some cases, coatings, envelopes, or protective matricescan be formulated to contain one or more of the polypeptides providedherein. Such coatings, envelopes, and protective matrices can be used tocoat indwelling devices such as stents, catheters, and peritonealdialysis tubing. In some cases, a polypeptide provided herein can beincorporated into a polymeric substances, liposomes, microemulsions,microparticles, nanoparticles, or waxes.

Nucleic Acids Encoding Polypeptides

This document also provides isolated nucleic acids that encode one ormore of the polypeptides provided herein. The term “isolated” as usedherein with reference to nucleic acid refers to a naturally-occurringnucleic acid that is not immediately contiguous with both of thesequences with which it is immediately contiguous (one on the 5′ end andone on the 3′ end) in the naturally-occurring genome of the organismfrom which it is derived. For example, an isolated nucleic acid can be,without limitation, a recombinant DNA molecule of any length, providedone of the nucleic acid sequences normally found immediately flankingthat recombinant DNA molecule in a naturally-occurring genome is removedor absent. Thus, an isolated nucleic acid includes, without limitation,a recombinant DNA that exists as a separate molecule (e.g., a cDNA or agenomic DNA fragment produced by PCR or restriction endonucleasetreatment) independent of other sequences as well as recombinant DNAthat is incorporated into a vector, an autonomously replicating plasmid,a virus (e.g., a retrovirus, adenovirus, or herpes virus), or into thegenomic DNA of a prokaryote or eukaryote. In addition, an isolatednucleic acid can include a recombinant DNA molecule that is part of ahybrid or fusion nucleic acid sequence.

The term “isolated” as used herein with reference to nucleic acid alsoincludes any non-naturally-occurring nucleic acid sincenon-naturally-occurring nucleic acid sequences are not found in natureand do not have immediately contiguous sequences in anaturally-occurring genome. For example, non-naturally-occurring nucleicacid such as an engineered nucleic acid is considered to be isolatednucleic acid. Engineered nucleic acid (e.g., a nucleic acid encoding apolypeptide comprising or consisting of the amino acid sequence setforth in SEQ ID NO:4, 6, or 8) can be made using common molecularcloning or chemical nucleic acid synthesis techniques. Isolatednon-naturally-occurring nucleic acid can be independent of othersequences, or incorporated into a vector, an autonomously replicatingplasmid, a virus (e.g., a retrovirus, adenovirus, or herpes virus), orthe genomic DNA of a prokaryote or eukaryote. In addition, anon-naturally-occurring nucleic acid can include a nucleic acid moleculethat is part of a hybrid or fusion nucleic acid sequence. A nucleic acidexisting among hundreds to millions of other nucleic acids within, forexample, cDNA libraries or genomic libraries, or gel slices containing agenomic DNA restriction digest, is not to be considered an isolatednucleic acid.

As used herein, the term “nucleic acid” refers to both RNA and DNA,including mRNA, cDNA, genomic DNA, synthetic (e.g., chemicallysynthesized) DNA, and nucleic acid analogs. The nucleic acid can bedouble-stranded or single-stranded, and where single-stranded, can bethe sense strand or the antisense strand. In addition, nucleic acid canbe circular or linear. Nucleic acid analogs can be modified at the basemoiety, sugar moiety, or phosphate backbone to improve, for example,stability, hybridization, or solubility of a nucleic acid. Modificationsat the base moiety include deoxyuridine for deoxythymidine, and5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidine fordeoxycytidine. Modifications of the sugar moiety can includemodification of the 2′ hydroxyl of the ribose sugar to form 2′-O-methylor 2′-O-allyl sugars. The deoxyribose phosphate backbone can be modifiedto produce morpholino nucleic acids, in which each base moiety is linkedto a six-membered, morpholino ring, or peptide nucleic acids, in whichthe deoxyphosphate backbone is replaced by a pseudopeptide backbone andthe four bases are retained. See, for example, Summerton and WellerAntisense Nucleic Acid Drug Dev., 7:187-195 (1997); and Hyrup et al.Bioorgan. Med. Chem., 4:5-23 (1996). In addition, the deoxyphosphatebackbone can be replaced with, for example, a phosphorothioate orphosphorodithioate backbone, a phosphoroamidite, or an alkylphosphotriester backbone.

A nucleic acid provided herein can comprise or consist of the sequenceset forth in SEQ ID NO:5.

Typically, an isolated nucleic acid provided herein is at least 10nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 50, 75, 100,200, 300, 350, 400, or more nucleotides in length). Nucleic acidmolecules that are less than full-length can be useful, for example, asprimers or probes for diagnostic purposes. Isolated nucleic acidmolecules can be produced by standard techniques, including, withoutlimitation, common molecular cloning and chemical nucleic acid synthesistechniques. For example, polymerase chain reaction (PCR) techniques canbe used. PCR refers to a procedure or technique in which target nucleicacids are enzymatically amplified. Sequence information from the ends ofthe region of interest or beyond typically is employed to designoligonucleotide primers that are identical in sequence to oppositestrands of the template to be amplified. PCR can be used to amplifyspecific sequences from DNA as well as RNA, including sequences fromtotal genomic DNA or total cellular RNA. Primers typically are 15 to 50nucleotides in length, but can range from 10 nucleotides to hundreds ofnucleotides in length. For example, a primer can be 12, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, or 45nucleotides in length. A primer can be purified from a restrictiondigest by conventional methods, or can be chemically synthesized.Primers typically are single-stranded for maximum efficiency inamplification, but a primer can be double-stranded. Double-strandedprimers are first denatured (e.g., treated with heat) to separate thestrands before use in amplification. General PCR techniques aredescribed, for example in PCR Primer: A Laboratory Manual, ed. byDieffenbach and Dveksler, Cold Spring Harbor Laboratory Press, 1995.When using RNA as a source of template, reverse transcriptase can beused to synthesize a complementary DNA (cDNA) strand. Ligase chainreaction, strand displacement amplification, self-sustained sequencereplication or nucleic acid sequence-based amplification also can beused to obtain isolated nucleic acids as described elsewhere (Lewis,Genetic Engineering News, 12(9):1 (1992); Guatelli et al., Proc. Natl.Acad. Sci. USA, 87:1874-1878 (1990); and Weiss, Science, 254:1292(1991)).

Isolated nucleic acids also can be chemically synthesized, either as asingle nucleic acid molecule (e.g., using automated DNA synthesis in the3′ to 5′ direction using phosphoramidite technology) or as a series ofoligonucleotides. For example, one or more pairs of longoligonucleotides (e.g., >100 nucleotides) can be synthesized thatcontain the desired sequence, with each pair containing a short segmentof complementarity (e.g., about 15 nucleotides) such that a duplex isformed when the oligonucleotide pair is annealed. DNA polymerase is usedto extend the oligonucleotides, resulting in a single, double-strandednucleic acid molecule per oligonucleotide pair, which then can beligated into a vector.

Isolated nucleic acids also can be obtained by mutagenesis. For example,a nucleic acid sequence encoding a polypeptide having the sequence setforth in SEQ ID NO:1, 2, 3, 4, 6, 7, or 8 can be mutated using standardtechniques such as, for example, oligonucleotide-directed mutagenesisand/or site-directed mutagenesis through PCR. See, Short Protocols inMolecular Biology, Chapter 8, Green Publishing Associates and John Wiley& Sons, Edited by Ausubel et al., 1992. Such mutations includeadditions, deletions, substitutions, and combinations thereof.

Vectors and Host Cells

This document also provides vectors containing a nucleic acid providedherein. As used herein, a “vector” is a replicon, such as a plasmid,phage, or cosmid, into which another DNA segment may be inserted so asto bring about the replication of the inserted segment. A vector can bean expression vector. An “expression vector” is a vector that includesone or more expression control sequences, and an “expression controlsequence” is a DNA sequence that controls and regulates thetranscription and/or translation of another DNA sequence.

In an expression vector provided herein, the nucleic acid can beoperably linked to one or more expression control sequences. As usedherein, “operably linked” means incorporated into a genetic construct sothat expression control sequences effectively control expression of acoding sequence of interest. Examples of expression control sequencesinclude promoters, enhancers, and transcription terminating regions. Apromoter is an expression control sequence composed of a region of a DNAmolecule, typically within 100 nucleotides upstream of the point atwhich transcription starts (generally near the initiation site for RNApolymerase II). To bring a coding sequence under the control of apromoter, it can be necessary to position the translation initiationsite of the translational reading frame of the polypeptide between oneand about fifty nucleotides downstream of the promoter. Enhancersprovide expression specificity in terms of time, location, and level.Unlike promoters, enhancers can function when located at variousdistances from the transcription site. An enhancer also can be locateddownstream from the transcription initiation site. A coding sequence is“operably linked” and “under the control” of expression controlsequences in a cell when RNA polymerase is able to transcribe the codingsequence into mRNA, which then can be translated into the polypeptideencoded by the coding sequence.

Suitable expression vectors include, without limitation, plasmids andviral vectors derived from, for example, bacteriophage, baculoviruses,tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses,poxviruses, adenoviruses, and adeno-associated viruses. Numerous vectorsand expression systems are commercially available from such corporationsas Novagen (Madison, Wis.), Clonetech (Palo Alto, Calif.), Stratagene(La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.).

An expression vector can include a tag sequence designed to facilitatesubsequent manipulation of the expressed nucleic acid sequence (e.g.,purification or localization). Tag sequences, such as green fluorescentprotein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc,hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequencestypically are expressed as a fusion with the encoded polypeptide. Suchtags can be inserted anywhere within the polypeptide including at eitherthe carboxyl or amino terminus.

This document also provides host cells containing a nucleic acidmolecule and/or nucleic acid vector provided herein. The term “hostcell” is intended to include prokaryotic and eukaryotic cells into whicha nucleic acid molecule or vector can be introduced. Any method can beused to introduce nucleic acid into a cell. For example, calciumphosphate precipitation, electroporation, heat shock, lipofection,microinjection, and viral-mediated nucleic acid transfer can be usedintroduce nucleic acid into cells. In addition, naked DNA can bedelivered directly to cells in vivo as described elsewhere (U.S. Pat.Nos. 5,580,859 and 5,589,466).

Detecting Polypeptides

This document provides methods and materials for detecting a polypeptideprovided herein. Such methods and materials can be used to monitorpolypeptide levels within a mammal receiving the polypeptide as atherapeutic. A polypeptide provided herein (e.g., an ABC-NP, ABC-NP1, orBC-NP2 polypeptide) can be detected, for example, immunologically usingone or more antibodies. As used herein, the term “antibody” includesintact molecules as well as fragments thereof that are capable ofbinding to an epitopic determinant of a polypeptide provided herein. Theterm “epitope” refers to an antigenic determinant on an antigen to whichthe paratope of an antibody binds. Epitopic determinants usually consistof chemically active surface groupings of molecules such as amino acidsor sugar side chains, and typically have specific three-dimensionalstructural characteristics, as well as specific charge characteristics.Epitopes generally have at least five contiguous amino acids (acontinuous epitope), or alternatively can be a set of noncontiguousamino acids that define a particular structure (e.g., a conformationalepitope). The term “antibody” includes polyclonal antibodies, monoclonalantibodies, humanized or chimeric antibodies, single chain Fv antibodyfragments, Fab fragments, and F(ab)₂ fragments. Polyclonal antibodiesare heterogeneous populations of antibody molecules that are containedin the sera of the immunized animals. Monoclonal antibodies arehomogeneous populations of antibodies to a particular epitope of anantigen.

Antibody fragments that have specific binding affinity for a polypeptideprovided herein (e.g., ABC-NP, ABC-NP1, or BC-NP2) can be generated byknown techniques. For example, F(ab′)2 fragments can be produced bypepsin digestion of the antibody molecule; Fab fragments can begenerated by reducing the disulfide bridges of F(ab′)2 fragments.Alternatively, Fab expression libraries can be constructed. See, forexample, Huse et al., Science, 246:1275 (1989). Once produced,antibodies or fragments thereof are tested for recognition of apolypeptide provided herein by standard immunoassay methods includingELISA techniques, radioimmunoassays, and Western blotting. See, ShortProtocols in Molecular Biology, Chapter 11, Green Publishing Associatesand John Wiley & Sons, Edited by Ausubel, F. M. et al., 1992.

In immunological assays, an antibody having specific binding affinityfor a polypeptide provided herein or a secondary antibody that binds tosuch an antibody can be labeled, either directly or indirectly. Suitablelabels include, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹I,³⁵S, ³H, ³²P, ³³P or ¹⁴C), fluorescent moieties (e.g., fluorescein,FITC, PerCP, rhodamine, or PE), luminescent moieties (e.g., Qdot™nanoparticles supplied by the Quantum Dot Corporation, Palo Alto,Calif.), compounds that absorb light of a defined wavelength, or enzymes(e.g., alkaline phosphatase or horseradish peroxidase). Antibodies canbe indirectly labeled by conjugation with biotin then detected withavidin or streptavidin labeled with a molecule described above. Methodsof detecting or quantifying a label depend on the nature of the labeland are known in the art. Examples of detectors include, withoutlimitation, x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers. Combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays.

Immunological assays for detecting a polypeptide provided herein can beperformed in a variety of known formats, including sandwich assays,competition assays (competitive RIA), or bridge immunoassays. See, forexample, U.S. Pat. Nos. 5,296,347; 4,233,402; 4,098,876; and 4,034,074.Methods of detecting a polypeptide provided herein generally includecontacting a biological sample with an antibody that binds to apolypeptide provided herein and detecting binding of the polypeptide tothe antibody. For example, an antibody having specific binding affinityfor a polypeptide provided herein can be immobilized on a solidsubstrate by any of a variety of methods known in the art and thenexposed to the biological sample. Binding of the polypeptide to theantibody on the solid substrate can be detected by exploiting thephenomenon of surface plasmon resonance, which results in a change inthe intensity of surface plasmon resonance upon binding that can bedetected qualitatively or quantitatively by an appropriate instrument,e.g., a Biacore apparatus (Biacore International AB, Rapsgatan, Sweden).Alternatively, the antibody is labeled and detected as described above.A standard curve using known quantities of a polypeptide provided hereincan be generated to aid in the quantitation of the levels of thepolypeptide.

In other embodiments, a “sandwich” assay in which a capture antibody isimmobilized on a solid substrate is used to detect the presence,absence, or level of a polypeptide provided herein. The solid substratecan be contacted with the biological sample such that any polypeptide ofinterest in the sample can bind to the immobilized antibody. Thepresence, absence, or level of the polypeptide bound to the antibody canbe determined using a “detection” antibody having specific bindingaffinity for the polypeptide. In some embodiments, a capture antibodycan be used that has binding affinity for BNP as well as a polypeptideprovided herein. In this embodiment, a detection antibody can be usedthat has specific binding affinity for a particular polypeptide providedherein. It is understood that in sandwich assays, the capture antibodyshould not bind to the same epitope (or range of epitopes in the case ofa polyclonal antibody) as the detection antibody. Thus, if a monoclonalantibody is used as a capture antibody, the detection antibody can beanother monoclonal antibody that binds to an epitope that is eithercompletely physically separated from or only partially overlaps with theepitope to which the capture monoclonal antibody binds, or a polyclonalantibody that binds to epitopes other than or in addition to that towhich the capture monoclonal antibody binds. If a polyclonal antibody isused as a capture antibody, the detection antibody can be either amonoclonal antibody that binds to an epitope that is either completelyphysically separated from or partially overlaps with any of the epitopesto which the capture polyclonal antibody binds, or a polyclonal antibodythat binds to epitopes other than or in addition to that to which thecapture polyclonal antibody binds. Sandwich assays can be performed assandwich ELISA assays, sandwich Western blotting assays, or sandwichimmunomagnetic detection assays.

Suitable solid substrates to which an antibody (e.g., a captureantibody) can be bound include, without limitation, microtiter plates,tubes, membranes such as nylon or nitrocellulose membranes, and beads orparticles (e.g., agarose, cellulose, glass, polystyrene, polyacrylamide,magnetic, or magnetizable beads or particles). Magnetic or magnetizableparticles can be particularly useful when an automated immunoassaysystem is used.

Antibodies having specific binding affinity for a polypeptide providedherein can be produced through standard methods. In general, apolypeptide can be recombinantly produced as described above, or can bepurified from a biological sample (e.g., a heterologous expressionsystem), and used to immunize host animals, including rabbits, chickens,mice, guinea pigs, or rats. For example, a polypeptide having the aminoacid sequence set forth in SEQ ID NO:4, 6, or 8 (or fragments thereofthat are at least six amino acids in length), can be used to immunize ananimal. Various adjuvants that can be used to increase the immunologicalresponse depend on the host species and include Freund's adjuvant(complete and incomplete), mineral gels such as aluminum hydroxide,surface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin anddinitrophenol. Monoclonal antibodies can be prepared using a polypeptideprovided herein and standard hybridoma technology. In particular,monoclonal antibodies can be obtained by any technique that provides forthe production of antibody molecules by continuous cell lines in culturesuch as described by Kohler et al., Nature, 256:495 (1975), the humanB-cell hybridoma technique (Kosbor et al., Immunology Today, 4:72(1983); Cole et al., Proc. Natl. Acad. Sci. USA, 80:2026 (1983)), andthe EBV-hybridoma technique (Cole et al., “Monoclonal Antibodies andCancer Therapy,” Alan R. Liss, Inc., pp. 77-96 (1983)). Such antibodiescan be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD,and any subclass thereof. The hybridoma producing the monoclonalantibodies can be cultivated in vitro and in vivo.

Alternative techniques for detecting a polypeptide provided hereininclude mass-spectrophotometric techniques such as electrosprayionization (ESI), and matrix-assisted laser desorption-ionization(MALDI). See, for example, Gevaert et al., Electrophoresis,22(9):1645-51 (2001); Chaurand et al., J. Am. Soc. Mass Spectrom.,10(2):91-103 (1999). Mass spectrometers useful for such applications areavailable from Applied Biosystems (Foster City, Calif.); BrukerDaltronics (Billerica, Mass.); and Amersham Pharmacia (Sunnyvale,Calif.).

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Biological Effects of ABC-NP, ABC-NP1, and BC-NP2Polypeptides

Polypeptides with the sequences set forth in FIGS. 1-3 were designed andsynthesized. The polypeptide set forth in FIG. 1 is referred to as anABC-NP polypeptide; the polypeptide set forth in FIG. 2 is referred toas an ABC-NP1 polypeptide; and the polypeptide set forth in FIG. 3 isreferred to as a BC-NP2 polypeptide. The biological effects ofintravenous ABC-NP, ABC-NP1, or BC-NP2 infusion were tested in normaldogs. Briefly, five normal dogs were infused with 25 μg of ABC-NP,ABC-NP1, or BC-NP2 per kg administered as an IV bolus. Urinary sodiumexcretion, urine flow, mean arterial blood pressure, plasma vasopressinlevels, and renal blood flow were measured as described elsewhere (Chenet al., Am. J. Physiol. Regul. Integr. Comp. Physiol., 288: R1093-R1097(2005) and Haber et al., J. Clin. Endocrinol. Metab., 29:1349-1355(2005)). The results were compared to results obtained from dogs treatedwith 25 μg of BNP per kg administered as an IV bolus.

Systemic administration of the ABC-NP polypeptide resulted in aquareticand natriuretic effects (FIGS. 4 and 5). Systemic administration of theABC-NP polypeptide had no effect on mean arterial blood pressure (FIG.6). Systemic administration of the ABC-NP polypeptide did not increaseplasma vasopressin levels (FIG. 7). Plasma vasopressin levels increasedfollowing administration of BNP (FIG. 7). Both ABC-NP and BNP resultedin a similar decrease cardiac filling pressures.

These results demonstrate that the ABC-NP polypeptide has distinct renaleffects and lacks the ability to affect systemic blood pressure ascompared to BNP.

Treatment with ABC-NP1 caused a reduction in mean arterial bloodpressure and an increase in renal blood flow (FIGS. 6 and 8). There wasno prolonged aquaretic and diuretic renal actions observed in dogstreated with ABC-NP1, which were observed in dogs treated with ABC-NP(FIGS. 4 and 5). Treatment with ABC-NP1 also indicated a strong trendfor increasing plasma vasopressin levels (FIG. 7). These resultsdemonstrate that the REA amino acid sequence (FIG. 1) in the ring of CNPis responsible for the lack of vasodilatory actions observed withABC-NP.

Treatment with BC-NP2 did not result in a decrease in mean arterialblood pressure or an increase in renal blood flow (FIGS. 6 and 8). Thus,like ABC-NP, BC-NP2 does not have vasodilatory effects. As compared toABC-NP, BC-NP2 only had about 50 percent of the renal effects (FIGS. 4and 5). These results demonstrate that the five amino acid residues ofANP present at the N-terminus of ABC-NP are involved in the prolongedaquaretic and diuretic actions of ABC-NP.

The biological effects of ABC-NP, ABC-NP1, or BC-NP2 on cardiacfibroblast cells were tested in vitro. Briefly, 80-90% confluent cardiacfibroblast cells were incubated in Hank's balanced salt solution(InVitrogen) containing 20 mmol/LN-[2-hydroxyethyl]piperazine-N′[2-ethanesulfonic acid], 0.1% bovineserum albumin, and 0.5 mmol/L 3-isobutyl-1-methylzanthine (Sigma).Treated cells received ANP (10⁻⁶ M), BNP (10⁻⁶ M), CNP (10⁻⁶ M), ABC-NP(10⁻¹¹ M, 10⁻⁸ M, or 10⁻⁶ M), ABC-NP1 (10⁻¹¹ M, 10⁻⁸ M, or 10⁻⁶ M), orBC-NP2 (10⁻¹¹ M, 10⁻⁸ M, or 10⁻⁶ M) for 10 minutes. Cells were lysed in6% TCA and sonicated for 10 minutes. The samples were ether extractedfour times in four volumes of ether, dried, and reconstituted in 300 μLcGMP assay buffer. The samples were assayed using a competitive RIA cGMPkit (Perkin-Elmer, Boston, Mass.). Briefly, samples and standards wereincubated with 100 μL anti-human cGMP polyclonal antibody andI¹²⁵-antigen for 18 hours. cGMP assay buffer was added to the samples,and they were centrifuged for 20 minutes at 2500 rpm. The free fractionwas aspirated off; the bound fraction was counted; and concentrationsdetermined Samples were corrected for dilution factors, and values wereexpressed as pmoles/mL. There is no cross-reactivity with ANP, BNP, orCNP, and there is <0.001% reactivity with cAMP, GMP, GDP, ATP, and GTP.

Treatment with ABC-NP1 activated cGMP in human cardiac fibroblasts,while treatment with ABC-NP and BC-NP2 did not (FIGS. 9-11). Theseresults demonstrate that ABC-NP1 has biological effect in human cardiacfibroblasts, while both ABC-NP and BC-NP2 do not appear to have anybiological effects in human cardiac fibroblasts. Thus, ABC-NP1 may havethe ability to induce anti-fibrotic actions, which may not be presentwith ABC-NP or ABC-NP2.

Example 2 Biological Effects of ABC-NP, ABC-NP1, and BC-NP2 PolypeptidesUsing Animal Models

The effects of ABC-NP, ABC-NP1, or BC-NP2 infusion is further assessedin two large animal models of sodium retention: the paced dog model ofCHF and a dog model of sodium retention which mimics cirrhosis andnephrosis. The first model is the rapid pacing model of CHF as describedelsewhere (Chen et al., Circulation, 100:2443-2448 (1999)). Briefly,dogs are paced at 240 bpm for 10 days to generate a model of severe CHF.Twenty-four dogs are paced, six receive ABC-NP polypeptides, six receiveABC-NP1 polypeptides, six receive BC-NP2 polypeptides, and six receivelasix (as diuretic control). Acute hemodynamic studies are performed atthe time of infusion and comparisons are made between groups and amongdogs at baseline and infusion. The second model is the TIVCC model ofsodium-retention and ascites without concurrent increases in cardiacfilling pressure as described elsewhere (Wei et al., Am. J. Physiol.,273:R838-844 (1997)). The ABC-NP, ABC-NP1, and BC-NP2 polypeptides aretested in each dog model of sodium-retention using increasing doses upto 100 pmol·kg/minute administered intravenously.

Example 3 Treating Patients at Risk of Left Ventricular Remodeling

Patients (e.g., myocardial infarction patients) at risk of leftventricular remodeling are treated with IV infusions of ABC-NP1polypeptide. Prior to the infusion, vital signs are taken and laboratorytests are performed to measure neurohormones and renal function. MRI isdone to measure left ventricular size and function. An intravenousinfusion of ABC-NP1 polypeptide is initiated at a dose of between 0.001μg/kg/minute and 1 μg/kg/minute. Vital signs are assessed every twohours during the infusion. The infusion can last for 72 hours. Blood isdrawn before the infusion is stopped at 72 hours to measureneurohormones and renal function. Patients return in one month and sixmonths for repeat MRI to assess left ventricular size and function.

Example 4 Treatment of Cardiorenal Conditions

Patients who develop worsening renal function with diuretic resistancein the setting of acute decompensated heart failure are treatedprospectively with IV infusions of ABC-NP or BC-NP polypeptides. Priorto the infusion, vital signs are taken and laboratory tests areperformed to measure electrolytes, serum creatinine, cystatin, and BNPpolypeptide levels. Baseline urine output is measured and urineelectrolytes are assessed. An intravenous infusion of ABC-NP or BC-NPpolypeptides is initiated. Vital signs and urine output are assessedevery 2 hours during the infusion, which is 12 to 72 hours in duration.Drug levels (e.g., ABC-NP or BC-NP polypeptide levels), BNP polypeptidelevels, serum creatinine, cystatin, and plasma and urine electrolytesare assessed daily throughout the infusion.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A natriuretic polypeptide less than 45 amino acidresidues in length, wherein said polypeptide comprises, in an order fromamino terminus to carboxy terminus: (a) the sequence set forth in SEQ IDNO:2, and (b) the sequence set forth in SEQ ID NO:3.
 2. The polypeptideof claim 1, wherein said polypeptide has natriuretic activity.
 3. Thepolypeptide of claim 1, wherein said polypeptide lacks vasodilatoryactivity.
 4. The polypeptide of claim 1, wherein said polypeptide lacksthe sequence set forth in SEQ ID NO:1.
 5. The polypeptide of claim 1,wherein said polypeptide comprises the sequence set forth in SEQ IDNO:8.
 6. The polypeptide of claim 1, wherein said polypeptide consistsof the sequence set forth in SEQ ID NO:8.
 7. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and anatriuretic polypeptide less than 45 amino acid residues in length,wherein said polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:2, and (b) thesequence set forth in SEQ ID NO:3.
 8. A method for increasing aquareticand natriuretic activity within a mammal without lowering bloodpressure, wherein said method comprises administering to said mammal anatriuretic polypeptide less than 45 amino acid residues in length,wherein said polypeptide comprises, in an order from amino terminus tocarboxy terminus: (a) the sequence set forth in SEQ ID NO:2, and (b) thesequence set forth in SEQ ID NO:3.
 9. The pharmaceutical composition ofclaim 7, wherein said polypeptide has natriuretic activity.
 10. Thepharmaceutical composition of claim 7, wherein said polypeptide lacksvasodilatory activity.
 11. The pharmaceutical composition of claim 7,wherein said polypeptide lacks the sequence set forth in SEQ ID NO:1.12. The pharmaceutical composition of claim 7, wherein said polypeptidecomprises the sequence set forth in SEQ ID NO:8.
 13. The pharmaceuticalcomposition of claim 7, wherein said polypeptide consists of thesequence set forth in SEQ ID NO:8.
 14. The method of claim 8, whereinsaid polypeptide lacks the sequence set forth in SEQ ID NO:1.
 15. Themethod of claim 8, wherein said polypeptide comprises the sequence setforth in SEQ ID NO:8.
 16. The method of claim 8, wherein saidpolypeptide consists of the sequence set forth in SEQ ID NO:8.